The long term goal of this research program is to achieve deep and clinically practical insights into the control of the heart, by molecular level investigation of the thin filament. This will be advanced by investigating the dynamic properties of the key regulatory proteins, cardiac troponin and tropomyosin, to reveal their modes of action and functions. The troponin- tropomyosin complex very tightly controls striated muscle contraction by conferring near- absolute calcium-dependence upon myosin's productive interactions with the thin filament. This regulation requires both flexibility and stiffness as important properties of troponin and tropomyosin. They must both move while attached to the thin filament in critical patterns, and also resist movement in other ways. Thus, the detailed dynamic properties of troponin and tropomyosin are particularly important for the regulation of cardiac contraction as well as having general significance for the function of these proteins. We have developed the means to study these dynamics with a very powerful approach: hydrogen/deuterium exchange with ultra high resolution mass spectrometry (HDX). The method holds great promise for understanding contractile regulation, and is uniquely available in our laboratory for this work. We will use HDX, strategically supplemented when valuable by electron microscopy, to ascertain critical aspects of troponin's regulatory switch mechanism. In one approach, we will investigate this mechanism by delineation of the effects of the troponin residues most essential for switching muscle contraction either on or off. Also, we will investigate troponin functions involving action at a distance by studies of troponin containing targeted mutations. Finally, we will determine and map the variable dynamics of tropomyosin that are vital for its function, using the same HDX approach. The overall aim of the work is to advance greatly our understandings of the basic molecular mechanism that directly controls the contraction of the heart and other striated muscles. PUBLIC HEALTH RELEVANCE: This research will provide molecular information on the control of muscle contraction generally, and the control of the heart's contraction in particular, by studying the intra-cellular protein machinery responsible for activating and inactivating each contraction. This subject is of fundamental importance for the causes and potential treatments of heart failure, coronary insufficiency, hypertension, and disorders of skeletal muscles.